The long-range objectives of this proposal are to identify the structural and biochemical properties of specific membrane domains and membrane-associated assemblies of mammalian spermatozoa and to determine how these membrane domains and membrane-associated assemblies function during the fertilization process. In the present proposal attention will be focused upon the membranes participating in the acrosome reaction, in order to identify mechanisms contributing to the membrane fusion process, and upon the plasma membrane of the postacrosomal segment, in order to characterize its role in gamete fusion. The specific objectives of this proposal are 1) to characterize the macromolecular complex (fuzzy-coat) which is exclusively localized on the luminal face of the outer acrosomal membrane; 2) to determine how the fuzzy-coat complex associates with integral membrane proteins of the outer acrosomal membrane; 3) to define the role of the fuzzy-coat complex in the membrane vesiculation process of the acrosome reaction; 4) to utilize antibodies against the fuzzy-coat complex to identify acrosome reacting sperm; 5) to determine if the fuzzy-coat assembly is modified during sperm maturation in the epididymis; 6) to identify enzyme activities associated with membrane domains participating in the acrosome reaction; 7) to characterize the postacrosomal sheath; and 8) to identify the intracellular distribution of cytoskeletal proteins in mammalian spermatozoa. Nitrogen cavitation and selective extraction procedures will be employed to disrupt spermatozoa and purified subcellular fractions consisting of specific membrane domains or membrane-associated assemblies will be isolated by a variety of centrifugation protocols. A combination of ultrastructural, biochemical and immunological techniques, which have previously been utilized to identify the functional properties of membrane-associated assemblies in other cell types, will be employed to pursue these specific aims. A resolution of these specific aims will contribute to our understanding of membrane physiology, to our understanding of major events in the fertilization process and to male reproductive biology.